The modular and programmable nature of DNA, coupled with its capacity for molecular recognition, positions DNA sensors as a uniquely versatile scaffold for molecular sensing within the cellular milieu. Sensors based on alternative scaffolds such as small molecule sensors Fluorescein and SNARF or GFP based pH sensors like pHluorin are significantly challenged in order to achieve simultaneous sensing of a specific analyte in multiple environments within the same cell. This has limited the study of fusion and fission events in biological systems e.g., endocytic sorting.
DNA has been molecularly chiselled to create a variety of intricate architectures on the nanoscale. Those DNA nano-architectures that can be chemically or physically triggered to switch between defined states, referred to as DNA sensors, present great potential for robotic and sensing applications on the nanoscale. Sub-cellular architectures are such nanoscale environments that offer rich possibilities to demonstrate functionality of these DNA nanoarchitectures.
The prior art achieves the mapping of intersecting endocytic pathways for receptors that (a) have cognate ligands (b) where the ligands are chemically functionalizable and (c) where such chemical functionalization of the ligand does not alter its trafficking characteristics. The vast majority of proteins that traffic via the plasma membrane do not satisfy these above criteria. In fact, several highly important trafficking membrane proteins do not fall into this category, and a full elucidation of their pathways has remained elusive. These pathways are now accessible using the targeting strategy described in the present disclosure.
The simultaneous functionality of multiple DNA sensors within the same cell still represents an outstanding challenge, the realisation of which would open up possibilities of multiplexed sensing and/or therapies in living systems. In order to realise this, the precise positioning of more than one DNA nanodevice within subcellular environments and demonstrating their simultaneous functionality therein is essential.
The present disclosure describes a technology called “SimpHony” (Simultaneous pH mapping Technology) based on DNA sensors, which demonstrates the simultaneous use of two or more DNA nanodevices within the same cell, each molecularly programmed to target a different cellular pathway and engineered to map pH gradients along the pathway. This technology is also called Multiplexing of DNA sensors.